Method of screening a gene

ABSTRACT

The present invention provides a method of screening genes based on expression information differing from gene expression information obtainable by DNA chip/DNA microarray techniques. The method of screening genes comprises performing in situ hybridization in respect of a tissue or cell sample from an organism using a probe which specifically hybridizes with mRNA and/or expression sequence tag being a product of gene expression, and examining localization of the mRNA and/or expression sequence tag in the tissue or cell.

This application claims priority benefit, under 35 U.S.C. §0 119(a), ofJapanese Patent Application No. 2001-112367, filed Apr. 11, 2001.

TECHNICAL FIELD

The present invention relates to a method of screening a gene.

BACKGROUND ART

Recently, a broad outline of human genome analysis has been publishedand the focus of research is shifting from genome analysis whichinvolves analysis of genome DNA sequence information, to expression(functional) analysis which involves analysis of gene expression. Atpresent, among expressed genes including expression sequence tags (EST),there are few whose function is understood even if the sequence thereofis already known.

In gene expression analysis, techniques for performing analysis withlarge samples at high speed and good efficiency (high throughputtechniques) are required. DNA chip/DNA microarray techniques can provideexpression information concerning several tens of thousands of genes inone cell, and are succeeding in effecting high throughput in geneexpression analysis experiments.

For example, by using DNA chip/DNA microarray techniques, it is possibleto identify genes, the expression level of which changes together withchanges in a disease condition. If the expression level of a particulargene correlates highly with the prognosis of a patient, then theexpression information concerning this gene can be used as an effectiveindicator in drug creation.

However, expression information obtainable by DNA chip/DNA microarraytechniques does not enable prediction of drug efficacy.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method of screeninggenes based on expression information which differs from gene expressioninformation obtainable from DNA chip/DNA microarray techniques.

Even if with DNA chip/DNA microarray techniques, the presence or absenceof expression, or level of expression of a gene in each cell can beunderstood, this information does not necessarily immediately lead todrug creation. The present inventors considered that it was possible toscreen a target from among numerous genes or expression sequence tagsthat have been cloned but whose functions are unknown, from the point ofview of localization of expression in tissue or cells of an organism.Further, using an in situ hybridization method, and by examininglocalization of expression in the tissues of an organism, the presentinventors have succeeded in establishing a system for screening a targetgene, thereby completing the present invention.

That is to say, the present invention provides a method for screening agene, by performing in situ hybridization with tissue of an organism, ora cell sample using a probe that specifically hybridizes with an mRNAand/or an expression sequence tag being a product of gene expression,and examining the localization of the mRNA and/or expression sequencetag in the tissue or cells.

By the method of the present invention, it is possible to screen targetsby not only screening genes which are structural units bearing geneticinformation but also expression sequence tags (EST) which do no morethan bear fragmentary genetic information.

In the method of the present invention, mRNA and/or expression sequencetags being the products of gene expression may be ones which express incultured cells or tissue. The mRNA and/or expression sequence tags beingthe products of gene expression can be those the expression of which hasbeen confirmed with a DNA chip or DNA microarray. Further, mRNA and/orexpression sequence tags being the products of gene expression, may beproducts, the expression level of which alters in response to an event.

Herein, an “event” refers to any kind of change occurring internally orexternally in an organism, and examples thereof include ischemia, tumor,and administration of a drug.

In the method of the present invention, a gene and/or expressionsequence tag which has been cloned but which is of unknown function canbe used.

Herein, “of unknown function” or “the function of which is unknown”refers to any of physico-chemical function, biochemical function orphysiological function having not been analyzed. Here, function on aphysicochemical level includes properties relating to intermolecularinteractions. For example, where a protein encoded by particular genebinds with DNA, the gene thereof can be said to encode a protein havinga function of binding with DNA (a function at a physico-chemical level).Function at a biochemical level includes properties involved inbiochemical processes. For example, a protein encoded by a particulargene binds with a gene promoter region within DNA, and it is clear thatthis activates transcription, the gene thereof can he said to encode atranscription activation factor (function on a biochemical level).Function at a physiological level included roles in organisms, tissuesor cells. For example, if a gene encoding a protein is disrupted, and amouse with no front legs is born, this protein can be said to have afunction involved in the differentiation of front legs (function at aphysiological level).

By a single screening according to the method of the present invention,the localization of at least two different types of mRNA and/orexpression sequence tag may be examined in one type of the same tissueor cells. By a single screening according to the method of the presentinvention, it is possible to examine localization of, for example, twoor more differing mRNA and/or expression sequence tags in the samesingle type of tissue or cell. For this, it is preferable to techniquefor performing a plurality of stainings simultaneously (double stain,triple stain, sky fish, etc.) by changing the fluorescence wavelength ofthe probe, and changing the secondary antibody.

Or, further, by a single screening according to the method of thepresent invention, localization of one type of mRNA or expressionsequence tag in at least two different types of tissue or cell may beexamined. By a single screening according to the method of the presentinvention, localization of one type of mRNA or expression sequence tagcan be examined in, for example, two or more types, preferably, 10 to 20types of different tissue or cell.

The method of the present invention can be used to screen for a geneencoding a substance effective as a medicament. For example, from thefact that expression of a gene is localizes at a specific site, byconsidering distribution within a tissue or cell, it is possible topredict the effect of that gene as a drug.

Further, the method of the present invention can be used to screen for agene related to a disease condition. For example, by using a diseasemodel animal, transgenic animal, knock-out animal or the like, it ispossible to more effectively select a useful probe by contrasting probelocalization and pathogenic site.

Further, the method of the present invention can be used to examine thefunction of a gene or expression sequence tag, which has been cloned butwhich is of unknown function. By knowing organ-specific localizationwithin cells or within tissues, it is possible to predict distributionof protein expression and probe targeting can be performed moreefficiently.

Further, the present invention provides a method of monitoring geneexpression which comprises collecting a tissue or cell sample from anorganism each before and after occurrence of an event, performing insitu hybridization in respect of the sample using a probe whichspecifically hybridizes with mRNA and/or expression sequence tag being aproduct of gene expression, and examining change in localization of themRNA and/or expression sequence tag in the tissue or cell. The tissue ofcell sample may be collected from an organism at at least two differentpoints in time after occurrence of an event.

The method of screening genes according to the present inventioninvolves the innovative approach of selecting a gene from the novelpoint of view of localization of a gene and/or expression sequence tagin the tissue or cells of an organism, and is useful in screening atarget gene.

Further, since the method of screening genes according to the presentinvention involves the selection of genes from the above-describedmorphologic point of view, it can lead research and development of drugsin a more correct direction. As a result, time and effort required fordrug, research and development can be reduced and costs can be lowered.

The method of monitoring gene expression of the present invention can beused in searching for a gene related to a condition, in searching forgene or EST which has been cloned but the function of which is unknown,in genome drug development, etc.

After genome wide screening, according to the method of the presentinvention, it is possible to screen a target gene by performing largescale screening from the point of view of localization in tissue orcells of an organism.

This will be explained in more detail by taking as an example the caseof a search for a drug useful in effecting recovery from damage due toischemia. In an ischemia model animal, a gene having an expression levelthat differs between before and after an event, being ischemia, isdetected with a DNA microarray or DNA chip (for example, thehigh-density oligonucleotide array GENECHIP™ (U.S. Affymetrics, Inc.))(genome-wide screening). Next, sequence information of the genes havingexpression levels that differ between before and after the ischemiaevent is obtained, by linking data obtained by DNA microarray or DNAchip with bioinformatics. Based on this sequence information, a probefor in situ hybridization is designed, and prepared. Thereafter, in situhybridization is used to examine how this gene is distributed in whattypes of tissue in an organ in which ischemia occurred (for example,brain, liver, etc.). In situ hybridization operations, can be eithermanual or automatic. For example, by using Ventana HX system (VentanaMedical Systems, Inc.) which realizes a complete automation of in situhybridization, results with good reproducibility can be obtained in ashort time. From the results of in situ hybridization, it is possible toscreen a gene which has a tissue distribution thought to be suitable forits use a drug. For example, where a drug for memory recovery is beingsought, a gene having expression localized in the hippocampus isselected. Further, where a drug which suppresses inflammation is beingsought, a gene the, expression of which is distributed in the entirebrain is selected.

Below, a mode for carrying out the present invention through combinationof gene expression analysis by DNA chip and in situ hybridization, willbe explained.

1. Gene Expression analysis by DNA chip

A method of analysis using conventional blotting techniques, whereinhybridization is performed simultaneously in respect of a plurality ofprobes arranged in an array, is generally referred to as arraytechnology (The chipping forecast. Nature Genetics, supplement vol. 21,(1999)). In particular, where an array is prepared with probes as spotsin an array form having a diameter of less that 1 mm, this is referredto as a microarray or chip, and arrays having probes constituted by DNAare referred to as “DNA chips”. At present, methods realizing this arraytechnology include a method where cDNA is spotted on a filter to form anarray, a method here cDNA or synthetic DNA is spotted on a slide glass,and further methods such as the hi-density oligonucleotide arrayGENECHIP™ (U.S. Affymetrics, Inc.) (Lockhart, D. J. et al. (1996),Expression monitoring by hybridization to high-density oligonucleotidearrays. Nature Biotechnology 14, 1675–80; Wodicka, L. et al. (1997)Genome-wide expression monitoring in Saccharomyces cerevisiae. NatureBiotechnology 15, 1359–67). In GENECHIPT™, spotting technology differsfrom that of other DNA chips. To distinguish between this and othermethods, this method per se is referred to by the trademark “GENECHIP™”.With GENECHIP™, it is possible to perform both genome analysis foranalyzing genome DNA mutation, and expression analysis for analyzinggene expression. However, in the present invention, expression analysisis performed.

1.1 Principle and Summary

In the GENECHIP™ technique, the method of spotting DNA probes on thechip differs from that of other DNA chips. In conventional methods, aDNA probe directly excised from an organism was spotted on a foundation.In contrast, with the GENECHIP™ technique, DNA is synthesized asfragments of 18 to 25 mer using a photochemical reaction in a stepresembling semiconductor manufacture techniques. As a result, severalmillion probes having 18 to 25 mer nucleotide sequences are immobilizedon a 50 or 24 μm square probe cell (this is referred to as a “tile”).

As a result of such differences in chip manufacture, properties such asthe following are produced:

(1) With a probe size of approximately 18 to 25 mer, there is aphenomenon where a mismatch probe having a single nucleotide substitutedin the middle of the probe will not readily hybridize (Goto, et al.(1997) Gene Diagnosis by Affinity Sensor BIACORE—theory andapplication—Clinical pathology 45, 224–28). Exploiting this, it ispossible to perform confirmation by perfect match and mismatch (probepair) for each probe arranged on each tile. By an operation such asthis, it is possible to eliminate false positive signals arising fromnon-specific: binding from the fluorescence intensity signals obtainedby the hybridization experiment, thus the system enables accuratemeasurement of signal strength arising from true probes having aperfectly matching nucleotide sequence (Lipshutz, R. J. et al. (1999)High density synthetic oligonucleotide arrays. Nature Geneticssupplement, volume 21, January)). Further, assisted by the shortness ofthe probe size and sample size, not only non-specific hybridization, butalso the background signal can be eliminated, thereby increasingquantitivity. Further, the synthesized oligonucleotide probe set isdetermined based on a coding region, a unique nucleotide sequence, andhybridization ability of the target gene.

Realized as a direct extension of these techniques is genome mutationanalysis. This method is a groundbreaking method involving detection ofa difference of a single nucleotide with p53, HIV, P450, SNP chip, etc.Since analysis is of a difference of one nucleotide sequence, a probecell having 4 to 5 probes corresponding to the four types of nucleotideG, A, T, C, for the site to be analyzed, and where necessary a probehaving this site deleted, is used.

(2) GENECHIP™ is constituted by a hybridization oven for binding thesample to the probes on the chip, a Fluidic Station for washing andlabeling, a Gene Array scanner for, reading fluorescence emissions, anda computer system for processing and analyzing the read information.Further., since experimental conditions from sample preparation to datacollection are optimized by using a pre-existing kit, it is possible toobtain data with high reproducibility. Since the expression levels ofseveral thousand genes are precisely assayed on a chip, arranging probesfor E. coli genes on the chip and mixing a fixed amount of cRNA derivedfrom E. coli genes in with the sample as a control (spiking) enablesquantitivity of the genes within the sample to be raised, as well asproviding a check on the precision of the operating process of theexperiment. Further, by using in conjunction with the results ofmeasurement using probes for housekeeping genes such as GAPDH and actin,comparative analysis of a plurality of different experimental resultscan be conducted, and reliable data having a wide dynamic range, can beobtained with high sensitivity without the user being troubled byexamination of experimental conditions, etc.

1.2 Items to be Prepared

GENECHIP™ is a comprehensive system encompassing steps from samplepreparation to data analysis, and is almost fully completed.Consequently, for reagents, kits, etc. to be used in each step, thefollowing, which are recommended by Affymetrics, Inc., are recommendedhere.

-   -   Isolation of total RNA        -   TRIzol Reagent (Gibco BRL Life Technologies)        -   RNeasy Total RNA Isolation Kit (QIAGEN)    -   Isolation of Poly(A)⁺ mRNA        -   Oligotex Direct mRNA Kit (QIAGEN)        -   Oligotex mRNA Kit (QIAGEN)    -   cDNA synthesis        -   Superscript Choice System (Gibco BRL Life Technologies)        -   T7-(dT)₂₄ Primer (GENSET Corp.)    -   Synthesis of Biotin labeled cRNA (In Vitro Transcription, IVT)        -   RNA Transcript Labeling Kit (Enzo)    -   IVT cRNA washing and quantification        -   RNeasy Mini Kit (QIAGEN)        -   CHROMA SPIN—100 columns (CLONTECH)    -   Buffer for fragmentation of labeled cRNA        -   200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc    -   Internal standard substance (Expression Control Clones)        -   pglbs-bioB, pglbs-bioC, pglbs-bioD, pglbs-cre

1.3 Protocol

The sequence of operations is as follows:

-   -   Step 1—Extract approximately 2 μg of Poly (A)* mRNA from the        sample.    -   Step 2—Perform cDNA synthesis with reverse transcriptase.    -   Step 3—Mass produce biotin-labeled cRNA by in vitro        transcription, and purify.    -   Step 4—Subject labeled cRNA to DNase treatment, or heat        treatment in the presence of magnesium ions thereby fragmenting        to a size of approximately 50 mer.    -   Step 5—After labeling a known internal standard (spiking), add        to sample, and pour onto Chip.    -   Step 6—Perform hybridization in an oven, and perform labeling in        a Fluidic Station.    -   Step 7—Import chip information with GeneArray Scanner.    -   Step 8—Perform data processing and analysis using a        Bioinformatics (biological information processing) system.

1.4 Bioinformatics

Since a large amount of data can be obtained with GENECHIP™, to use itefficiently, so-called bioinformatics (Bioinformatics) techniques arerequired. For this purpose, in GENECHIP™, as bioinformatics tools, theproprietary GENECHIP™ Laboratory Information Management System (LIMS™)and GENECHIP™ Expression Data Mining Tool (EDMT™) are providedtherewith, and these enable data to be input into a SQL compliantdatabase in a format determined by an open consortium for standardizinggene-related analysis techniques (GATC), and linked to gene informationdatabases (GenBank, etc.) published on the internet. However, sincebioinformatics per se is still in a developmental stage, there are caseswhere data analysis with known systems is insufficient. In small andmedium scale research facilities, there arises the need to separatelyfile and analyze the databases of a few individuals, and use otheranalysis programs to perform data processing, graphing, and statisticalcalculations. Here, the present inventors naturally access an LIMS-SQLserver and use an EDMT-like tool to process data, store individual datain a GATC compatible extension database, and using Gene Spring (U.S.Silicon Genetics, Inc.) perform clustering, tabulation, searches, andinformation database searches. Further for statistical calculations, andanalysis of the functional hierarchy of individual genes, the presentinventors use Stingray (U.S. Affymetrics, Inc.).

2. Examination of Localization of Expressed Genes by In SituHybridization

The present inventors performed in situ hybridization with fresh frozenslices. Fresh frozen slices have the strength that compared with othertissue samples such as paraffin immobilized (issue and tissue embeddedafter immobilization, a signal can most easily be obtained. This isbecause permeability into the probe tissue is high.

Table 1 shows a flow chart for in situ hybridization using a freshfrozen sample. The process is explained below in accordance with theflow chart.

TABLE 1 Flow chart of in situ hybridization using Fresh Frozen Sample

2.1 Preparation of Fresh Frozen Slices

Since a fresh frozen slice is a raw sample, it is easily compromised byRNase, and to proceed with the experiment, it is necessary to performthe experiment in as near an RNase-free state as possible, andparticular attention must be made to preserving the mRNA in the tissue.

(1) Analyte

A sample (e.g. brain or liver) is excised from an experimental animal.Gauze that has been soaked in physiological saline solution is placed ona clean sterilized plate, and the sample is wrapped to prevent drying.Further, where there is a thick coat of film or where venous tissue orinterstitial tissue is included, since it is difficult to obtain thinslices, these are preferably cut away.

(2) Embedding tissue

A small amount of OCT compound (manufactured by Milles Lab) is placed atthe bottom of a plastic vessel for embedding, or of aluminum foil moldedinto a cylinder. Tissue cut to an appropriate size is placed thereon,and OCT compound is injected from above such that the tissue comes tothe center. These are then immersed in acetone which was previous placedin dry ice, to quick freeze. After freezing, these are transferred to aplate containing dry ice, and after embedding of the tissue has beencompleted, stored in a freezer at −80° C.

(3) Thin slicing of tissue

Slices, 10 μm in size, are cut using a cryostat. After cutting, slicesare adhered to 3-aminopropyl triethoxy silane (ASP)-coated slideglasses. A method of preparing an ASP-coated slide glass is shown inTable 2.

TABLE 2 Method of Preparing APS coated slide glass 1. Dissolve APS(Sigma) in acetone to 1 to 2%. 2. Place slide glass in rack, and immersein APS solution for 5 to 10 seconds. 3. Wash gently with acetone. 4.Wash with DEPC processed distilled water* 5. Air dry overnight within ahood Note: *DEPC processed distilled water: Solution in which RNase hasbeen inactivated, which is prepared by adding 0.1% DEPC(diethylpyrocarbonate) to distilled water, stirring well in a stirrer,and placing in an autoclave after allowing to stand overnight.

After tissue is adhered to the slide glass, it is immediately driedusing cooled air.

(4) Preservation of tissue

After slicing thinly, immobilization is performed using 4%paraformaldehyde. The in situ hybridization step may be performedimmediately thereafter. If in situ hybridization is not performedimmediately, the slice is dried for 30 minutes or more with cooled air,placed in a slide glass rack, scaled with vinyl tape, and stored at −80°C.

With extra tissue remaining after slicing, an OCT compound is placed onthe cut face thereof, processing to avoid drying of the tissue isperformed, and the tissue is stored at −80° C.

(5) Examination of remaining RNA in the tissue

No matter what kind of tissue, mRNA within the tissue will be destroyedby the various operations, and evaluation of the remaining RNA isimportant. Methods for evaluation are broadly divided into specialstaining methods and in situ hybridization methods. A special stainingmethod involves staining the whole of the RNA including mRNA withacridine orange or methyl green/pyroline Y stain. Methods for evaluatingremaining RNA with in situ hybridization, involve performing in situhybridization in respect of β-actin, poly-A RNA, and 28 S ribosome RNA(rRNA). In the case where RNA staining provides a negative result, theRNA is judged not to be present in the tissue, and thus the tissue isunsuitable for in situ hybridization. On the other hand, where apositive result is obtained by RNA staining, unless the in situhybridization treatment is performed properly, a positive finding cannotbe obtained. For example, cases are known where the probe cannot reachthe target mRNA. Therefore, a positive control is essential for in situhybridization. β-actin is used as an internal control in procedures suchas Northern blot, however, its level often varies according toconditions such as cell proliferation. A probe for performing in situhybridization with the poly A of mRNA has, in comparison to a normalprobe, a totally different GC-content, and the Tm value differs, so itbecomes necessary to change the in situ hybridization conditions. As aresult, this hybridization cannot be performed simultaneously withnormal in situ hybridization, and is therefore not generally employed.In contrast, 28S rRNA is distributed widely in all cells, and itsproduction level is very constant. Further, with the 28S rRNA oligoprobe employed by Yoshii, et al. (Yoshii A, et al., J HistochemCytochem, 43:321–327, 1995), it is possible to use the same probe indifferent species enabling in situ hybridization to be performed withthe same probe on experimental model animals to humans.

2.2 In situ hybridization

Below, processing of the probe and slice will be explained.

(1) Preparation of probe and labeling

(1-1) Preparation of probe

As a probe, a double-stranded DNA (dsDNA), oligonucleotide(approximately 20 to 40 nucleotides in length), or RNA probe can beused.

To prepare an RNA probe, it is preferable to prepare a template, performin vitro transcription, and confirm the purity and concentration of theRNA probe.

In the preparation of templates, there are the following cases: (a) acase where a plasmid is adopted as a template, (b) a case where a PCRproduct from a plasmid is adopted as a template, and (c) a case where aPCR product from cDNA is adopted as a template. Case (a) where a plasmidis adopted as a template is most common, however, the present inventorshave been successful with the method of case (c). In the method of case(a), first, a probe DNA fragment is incorporated into a plasmid havingpromoters such as SP6, T3, T7, etc. (approximately 3 to 4 weeks).Plasmid DNA is cleaved with restriction enzymes and linearized. Next, tomake it RNase-free, the linearized plasmid DNA is processed withproteinase K. In the method of case (c), mRNA is extracted from frozentissue, and cDNA is synthesized. PCR is performed (approx. 7 to 10 days)using primers including an RNA polymerase promoter sequence, upstream ordownstream thereof (since both antisense and sense probes are prepared).For example, a T7 promoter sequence can be incorporated at the 5′terminus of the 3′-side primer for preparation of the antisense probe,and a T7 promoter sequence may be incorporated at the 5′ terminus of the5′-side primer for preparation of the sense probe.

In vitro transcription can be performed using a commercially availablekit (e.g. AmpliScribe™ T7 Transcription Kit (EPICENTRE TECHNOLOGIES)).

Purification of the reaction product (RNA probe) can be performed usinga commercially available kit (e.g. RNeasy minikit (QIAGEN)).

Absorbance of the purified reaction product is measured and RNAconcentration calculated.

(1-2) Probe labeling

Labeling techniques include radioactive labeling anti non-radioactivelabeling. In radioactive labeling 35S is widely used. Non-radioactivelabeling techniques include methods of labeling with digoxigenin whichis a hapten, or biotin, etc. and the T—T dimer method which involvesformation of dimers of thymine which is a nucleotide of nucleic acids byirradiation with UV (Koji T, et al. Acta Pathol Jpn, 40: 793–807, 1990).

Hapten labeling can be easily performed using a commercially availablekit. In the case of a dsDNA probe, this can be labeled with digoxigeninby using a random primer method (e.g. DIG DNA labeling Kit manufacturedby Boehringer). An oligonucleotide probe can be digoxigenin labeledeasily using DNA Tailing Kit (manufactured by Boehringer) and an RNAprobe can be digoxigenin labeled easily using a DIG-RNA Labeling Kit(manufactured by Boehringer).

Whether or not labeling was effected, can be examined by developing on amembrane. That is, the labeled probe is step-wise diluted by factors of10, to prepare an approx. 10 ng/μl to 1 pg/ μl solution. This is drippedonto nylon (cellulose) membrane, 1μ1 at a time and allowed to dry.Thereafter, it is allowed to develop using a method that will allowactual development in situ hybridization (using alkali-phosphatase orperoxidase-labeled anti-hapten antibody). If the label is sufficientthen the label should develop with good sensitivity. In the case wherethere is no development, or where sensitivity is poor, this means thatthe label is insufficient. In this case, since a kit contains an alreadylabeled positive control, this is confirmed with simultaneous staining.Further, a digoxigenin assay strip (Boehringer 1669958) is commerciallyavailable with which it is possible to simply check whether or not theprobe has been well labeled. Biotin and digoxigenin are frequently usedas haptens for labeling. The kidney, liver, and muscle, etc. includelarge amounts of endogenous biotin, and where fresh frozen tissue isused, considering background after staining, digoxigenin is the morepreferable.

Further, in addition to examining whether or not probes have beenlabeled, whether or not hybridization is properly occurring amongsense-antisense nucleic acids is examined by performing dothybridization on a membrane. First, a serial dilution of unlabeled senseprobe is prepared, dripped onto a nylon membrane and allowed to dry.This is then allowed to hybridize with a labeled antisense probe andthereafter, developed using the enzyme antibody method in the samemanner as described in the below-described in situ hybridization withtissue.

(1-3) Tissue processing and hybridization

1. Rehydration

Where slices which had been adhered to ASP coated slide glasses werebeing stored at −80° C., after removing the slide glass box containingthe slices from the freezer, the slide glass box is placed whileremaining sealed in a 37° C. heater, and opened after warming forapprox. 60 minutes. After placing in a slide glass rack, the slides aresoaked in PBS for 3 minutes to rehydrate.

2. Immobilization

Tissue slices are immobilized in 4% paraformaldehyde/PBS solution forapprox. 15 minutes at room temperature. After immobilization, tissueslices are placed in a staining vat containing PBS, a while shaking on ashaker are wash for 3 minutes, 3 times.

3. Protein removal

With unprocessed tissue, since probe permeability is insufficient,protein removal treatment of the tissue is required. In the protocolhere, protein removal is performed by hydrochloric acid and proteinaseK. First, as a hydrochloric acid treatment, tissue is immersed in astaining vat containing 0.2N HCl (diluted with distilled water), for 10to 20 minutes, at room temperature. Thereafter, PBS washing is performedfor 3 minutes, 3 times, while shaking on a shaker. Next, proteinase K(molecular biology use) treatment is performed. A solution having itfinal proteinase K concentration of 0.5 μg/ml/PBS is prepared, andpreviously left in a 37° C. water bath for 30 minutes. The solution isallowed to penetrate into the slice at 37° C. for 5 to 15 minutes, andthereafter the slice is washed with PBS for 3 minutes, 3 times on theshaker.

4. Post-immobilization

Tissue loosened by protein removal is tightened. In particular, in thecase of fresh frozen slices, this is necessary from preservation offorth. Tissue slices are immobilized in 4% paraformaldehyde/PBS for 5 to10 minutes at room temperature. Note that after post-immobilization,slices are washed with PBS to neutralize aldehyde remaining in thesample, and may be immersed twice in 2 mg/ml glycine/PBS for 15 minutes.Thereafter washing is performed.

5. Pre-hybridization

Tissue slices are immersed in pre-hybridization solution(4×SSC/3×Denhardt's solution/20% formamide) at room temperature for 30minutes (slices may be immersed for in excess of 1 hours). By performingthis processing, the hybridization solution takes to the slices moreeasily.

6. Hybridization

A hybridization solution (4×SSC/50 mM sodium phosphate buffer solution(pH 8.0)/5×Denhardt's solution/0.2 mg/ml salmon sperm DNA/0.2 mg/mlyeast tRNA/20% formamide/labeled probe) is prepared, quenched afterboiling and then placed on the tissue slice.

Probe concentration differs according to the tissue, probe, and targetgene but is typically used in a range of between 0.5 to 2 ng/μl.

In the case of a dsDNA probe, hybridization solution/probe is boiled.This is to eliminate the secondary structure of the probe. In the caseof an oligonucleotide probe, hybridization solution/probe is preferablyboiled. Specifically, the lid of a 1.5 ml Eppendorf tube containing thehybridization solution is closed, the tube placed in a stainless steelboiling vessel, the lid closed, and boiling performed for 5 to 7minutes. Thereafter, the Eppendorf tube is placed directly into a vesselcontaining ice and water to quench. Then, the Eppendorf tube is placedon ice.

The slide glasses soaked in pre-hybridization fluid is taken out andexcess solution around the tissue is wiped off. At this time, care istaken that the tissue does not dry out. Tissue drying is a cause ofnon-specific signals. Approx. 30 to 70 μl of hybridization solution isthen placed on each slide glass and stirred well.

Thereafter, slide glasses on which the hybridization solution was placedare placed in a humid box. A cover is placed thereon to avoid drying,and sealed with vinyl tape, and then it is allowed to stand over nightat 37° C.

7. Post-hybridization washing

From the sealed humid box, slides are removed one by one, and placed ina staining vat which contains wash solution. The slides are washed withthe following wash solution for 30 minutes each 2 times on a shaker.

Wash solution

-   -   2×SSC+0.075% Briji35 (23 Lauryl ether, Sigma)    -   0.5×SSC+0.075% Briji35        (1-4) Visualization of hybridized probe

In the case of a radioactive label, autoradiography is performed. In thecase of a non-radioactive label, an enzyme-antibody method (using ananti-digoxigenin antibody or anti-biotin antibody) probes whichhybridize with a target gene (mRNA, DNA) are allowed to develop. Here,detection of a signal according to enzyme antibody methods will beexplained.

In the case of labeling with digoxigenin, an anti-digoxigenin antibodyis used, but in this case, there are the following three methods: (a) amethod using a peroxidase labeled anti-digoxigenin antibody, (b) amethod using an alkali phosphatase labeled anti-digoxigenin antibody,and (c) a method using a mouse anti-digoxigenin antibody. Methods (a)and (b) are direct methods, and method (c) is an indirect method furthercombining a peroxidase- or alkali phosphatase-labeled antibody. Amongthese, the method with the highest sensitivity is method (b), and themethod with the lowest sensitivity is method (a). Even with method (c)it is possible to obtain good sensitivity.

Steps hereafter are the same as normal immunochemical staining and RNaseis of no particular concern.

First, pre-hybridization is performed in order to prevent non-specificbinding to tissues of the anti-digoxigenin antibody. The pre incubationsolution used at this time includes bovine serum albumin, an equivalentscrum in which a primary antibody is prepared, or IgG (if a rabbitantibody, then normal rabbit serum and IgG). Development is usuallyperformed with diaminobenzedine (DAB) and hydrogen peroxide, however,there exist various methods for increasing sensitivity even at thisstage. For example, it is possible to increase sensitivity by addingCoCl₂ and NiSO₄(NH₄)₂SO₄. Further, recently, a kit called CatalyzedSignal Amplification (CSA system, DAKO) has become commercial available,and the sensitivity thereof is extremely good. After developing, nucleusstaining is performed. Nucleus staining can be abbreviated where imageanalysis is performed however it is preferable for clarifying signalnegative cells. Where DAB is used as a development pigment, sincedevelopment with DAB is usually brown, it is preferable to use methylgreen staining for nucleus staining since it will be easily visualized.

Table 3 shows the operating steps where a peroxidase labeledanti-digoxigenin antibody is used.

TABLE 3 Post-Hybridization Development: Case where a Peroxidase-labeledAnti-digoxigenin Antibody is used. 1. On the day followinghybridization, a methanol block for blocking wash solution, peroxidasewithin tissue. Immerse in methonal + 0.3% hydrogen peroxide for 20 minat room temperature. Shut out light with aluminum foil. Wash with PBS.2. Pre-incubation (normal sheep IgG/BSA/PBS) in a humid box, roomtemperature for over 30 min. 3. Dilute peroxidase labeled sheepanti-digoxigenin antibody with pre- incubation solution 50 to 200 times,and drip onto tissue slices. React for over 1 hour, to overnight, atroom temperature in a humid box. 4. Wash with 0.075% Brij 35/PBS. 5 min.4 times. 5. React with DAB, hydrogen peroxide, stain nucleus, dehydrateand encapsulate

When an alkali phosphatase-labeled anti-digoxigenin antibody is used,there are fewer steps and good sensitivity can be obtained. Table 4shows the operating steps where an alkali phosphatase labeledanti-digoxigenin antibody is used.

TABLE 4 Post-hybridization Development: Where an AlkaliPhosphatase-labeled Anti-digoxigenin Antibody is used. 1. Afterhybridization, wash and then place in buffer solution 1*¹. 5 min. 2.Pre-incubation: 1 to 1.5% blocking solution/buffer solution 1, 1 hour.3. Place alkali phosphatase labeled anti-digoxigenin antibody dilutedwith buffer solution 1, by a factor of 500 to 2,000 on tissue, andreact. 30 min to 1 hr. 4. Wash: 15 to 30 min with buffer solution 1,twice, on the shaker. 5. Buffer Solution 2*² for 3 min. 6. React withdeveloping solution (NBT 6 μl/ml + BCIP 3.5 μl/ml + levamisole*³/buffersolution 21 ml). Watch level of development, and stop reaction*⁴. 7.Wash with water, dehydrate, and encapsulate. Where this kit is used,there is little loss of color even after washing, dehydrating andencapsulating. Where another method of developing alkali phosphatase isused, color will easily be lost by washing and dehydrating, so afterwashing gently, and sponging off water on the tissue, allow to dry, andencapsulate with a water-soluble encapsulating agent. *¹100 mM Tris HClpH 7.5, 150 mM NaCl *²100 mM Tris HCl pH 9.5, 100 mM NaCl, 50 mM MgCl₂*³Used to stop non-specific alkali phosphatase development on thetissue. Pre-dissolved solutions such as the one produced by DAKO areavailable. *⁴Actual reaction time varies from 5 minutes to around 12hours.

Here, development with nitro blue tetrazolium (NBT) and5-bromo-4-chloro-3-indolyl phosphate (BCIP) is indicated. The DIGNucleic Acid Detection Kit manufactured by Boehringer is convenient.

For reference, the above operations are described in “In situhybridization techniques”, Takehiko Shoji (ed.), Gakusai Kikaku.

In situ hybridization may be performed using Ventana HX system(manufactured by Ventana, Inc.) which realizes full automation. By usingthis device, it is possible to a large quantity of results with goodreproducibility in a short period.

By the above operations, it is possible to examine the state oflocalization of mRNA expression at the tissue level or at the celllevel.

Gene expression localization information obtained as above can be usedin searching for a gene related to a condition, in searching for gene orEST which has been cloned but the functions of which is unknown, ingenome drug development, etc.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows change in expression level in hippocampus of various genesand expression sequence tags after 24 hours had passed since recovery ofblood flow in a brain ischemia model rat.

FIG. 2 shows change in expression level in liver of various genes andexpression sequence tags after 4 hours had passed since recovery ofblood flow in a hepatic ischemia model rat.

FIG. 3 shows results of analysis by in situ hybridization ofdistribution of Hsc70 expression in liver tissue of a control rat.

FIG. 4 shows results of analysis by in situ hybridization ofdistribution of Hsc70 expression in liver tissue after 4 hours hadpassed since recovery of blood flow in a hepatic ischemia model rat.

FIG. 5 shows results of analysis by in situ hybridization ofdistribution of TATase expression in liver tissue of a control rat.

FIG. 6 shows results of analysis by in situ hybridization ofdistribution of TATase expression in liver tissue after 4 hours hadpassed since recovery of blood flow in a hepatic ischemia model rat.

FIG. 7 shows change in expression levels of various genes and expressionsequence tags in the brain of a brain ischemia model rat after 2 hourshad passed since recovery of blood flow. Expression levels of HSC70,HSP70, c-jun, EST1 and EST2 are clearly shown.

FIG. 8 shows the results of analysis by in situ hybridization ofdistribution of HSP70 expression in brain tissue of a control rat andbrain tissue after 2 hours had passed from recovery of blood flow in abrain ischemia model rat.

FIG. 9 shows the results of analysis by in situ hybridization ofdistribution of c-jun expression in brain tissue of a control rat andbrain tissue after 2 hours had passed from recovery of blood flow in abrain ischemia model rat.

FIG. 10 shows the results of analysis by in situ hybridization ofdistribution of EST1 expression in brain tissue of a control rat andbrain tissue after 2 hours had passed from recovery of blood flow in abrain ischemia model rat.

FIG. 11 shows magnified views (x250, x500) of FIG. 10.

FIG. 12 shows the results of analysis by in situ hybridization ofdistribution of EST2 expression in brain tissue of a control rat andbrain tissue after 2 hours had passed from recovery of blood flow in abrain ischemia model rat.

BEST MODE FOR CARRYING OUT THE INVENTION

Below, the present invention is explained in detail by use of examples.These Examples are provided to explain the present invention, but not tolimit the scope of the present invention

EXAMPLE 1

An adult mouse (male, Bcl black, purchased from Sankyo Lab Services)whose common carotid arteries were bilaterally ligated for 20 minutes tointerrupt blood flow was adopted as a brain ischemia model. Thereafter,mice were euthanized after passage of time from recovery of blood flow(0 to 24 hours), the hippocampus removed, and a sample preparedaccording to the protocol. Gene expression analysis was performed usinga Mu6,500 Oligonucleotide DNA Probe array with the high-densityoligonucleotide array GENECHIP™ system of Affymetrics, Inc. (U.S.). WithMu6,500 Oligonucleotide DNA Probe array, 6500 types of genome could beanalyzed simultaneously. Table 1 shows results of analysis usingbioinformatics (specifically, a scatter plot by LIMS-EDMT was used) onthe basis of this data. The horizontal axis of FIG. 1 shows the genomeexpression level of a rat (control) in which ischemia processing was notconducted. The vertical axis indicates the genome expression level 24hours after ischemia/reperfusion. The individual points in FIG. 1correspond to respective specified genomes. It was possible todifferentiate between those where expression level had increased (e.g.,in *1, there was an increase in expression of approx. 20 times from 30to 600 as between before and after ischemia), those for which there wasno change (for example, *2), and those which were reduced (for example,in *3, there was a decrease in expression of a factor of approx. 1/100from 7,000 to 70 as between before and after ischemia, and thus we wereable to know in general terms about the expression level in the tissueof specific genes. This result confirmed changes in expression level ofapproximately 1,000 types of genes. If this is connected with publiclyavailable gene information databases, individual genome information canbe instantaneously obtained.

EXAMPLE 2

An adult rat (male 12-weeks old, Wister-type male rat purchased fromSankyo Lab Services) in which the hepatoportal portion was ligated for15 minutes to interrupt blood flow, was adopted as a hepatic ischemiamodel. Thereafter, rats were euthanized after passage of time (0 to 4hours) after recovery of blood flow, the liver removed and samplesprepared according to the protocol. Gene expression analysis wasperformed using a Rat Toxicology U34 array with the hi-densityoligonucleotide array GENECHIP™ system of Affymetrics, Inc. (U.S.). Withthe Rat Toxicology U34 array, approximately 850 types of rat gene andEST could be analyzed simultaneously. FIG. 2 shows results of analysisusing bioinformatics (specifically, a scatter plot by LIMS-EDMT wasused) on the basis of this data. The vertical axis of FIG. 2 shows thegenome expression level of a rat (control) in which ischemia processingwas not conducted. The horizontal axis indicates the genome expressionlevel 4 hours after ischemia/reperfusion. The individual points in FIG.2 correspond to respective specified genomes. Examples of these includeHsc70 and TATase (Tyrosine aminotransferase). As a result, comparingbetween 0 hours and 4 hours, there were approximately 100 types wheregene expression had increase 2 times or more and approximately 40 typeswhere gene expression had fallen to ½ or less. In respect of Hsc70 andTATase, there was no dominant change in expression level according toGENECHIP™ was exhibited in respect of both genes as between the controlgroup and the ischemia treated.

EXAMPLE 3

Livers were removed respectively from a control rat and the hepaticischemia model rat (after 4 hours had passed since recovery of bloodflow) of Example 2 and fresh frozen slices were prepared. UsingDigoxigenin-labeled position 229–629 (400 bp) of HSC70 (Neat shockprotein 70-like protein, NCBI GenBank Accession No. M11942) as an RNAprobe, in situ hybridization of the fresh frozen slices was performedwith Ventana HX system. Primer sequences used in preparation of RNAprobes are shown in Table 5 and in situ hybridization conditions areshown in Table 6.

TABLE 5 No. Upper Primer Position Lower Primer Position Length HSC70(heat shock protein 70 like protein) M11942 CAATGAACCCCACCAACACAG 229CTTTCAGCCCCGACTTCTTA 629 400 bp (SEQ ID NO:1) (SEQ ID NO:2) HSP70 L16764GCTGGTGGGCGGCTCGAC 1182 GCTCTTGTCCGTGGCCGTGAC 1659 478 bp (SEQ ID NO:3)(SEQ ID NO:4) TATase X02741 GAAGAAAGAAAGGCAGGAAGG 192CTTGGAATGAGGATGTTTTGT 594 403 bp (SEQ ID NO:5) (SEQ ID NO:6) c-junX17163 TGAAGCAGAGCATGACCTTG 453 AGTTGCTGAGGTTGGCGTAG 878 426 bp (SEQ IDNO:7) (SEQ ID NO:8) EST 1 AA818604 GCGATCTCCTTCATCTTGGT 147GACTTGGGCACCACCTACTC 511 365 bp (SEQ ID NO:9) (SEQ ID NO:10) EST 2A1103915 TGGGCTCAAAGCCATATTTC 183 CCGAACTCTAGAGCCACCAG 585 403 bp (SEQID NO:11) (SEQ ID NO:12)

TABLE 6 Step Reagent Temp. Time Off line Fixation  4% PFA/PBS R.T.  30min Wash PBS R.T.   5 min × 2 times DEPC treatment  0.1% DEPC/PBS R.T. 15 min × 2 times Wash PBS R.T.   1 min H₂O R.T.   1 min Acid treatment 0.2M HCl R.T.  20 min Wash H₂O R.T.   1 min PBS R.T.   3 min × 3 times 5_(×) SSC R.T.  30 min Probe App. 200 μL  50% FA, 5_(×) SSC,  5_(×)Denhardt's, 500 ug/ml ssDNA, 250 ug/ml t-RNA, 1 mM DTT Denaturation 65°C.  15 min Hybridization 57° C.  14 hrs Stringency Wash 1^(st)–2^(nd)  2× SCC 55° C.   6 min × 2 times 3^(rd)–4^(th)  0.1 × SSC 55° C.  16 min ×2 times Antibody Blocking Protein Brock 37° C.  20 min AntibodySerum-Free × Wash 2000 anti DIG-AP 37° C.  46 min TBS R.T  10 min × 3times APB R.T   5 min Detection BM-Purple R.T   6 hrs~

Results for the control rat are shown in FIG. 3, and results for thehepatic ischemia model rat are shown in FIG. 4. Within the figures,Anti-sense shows the results of staining with antisense probe, and Senseindicates results of staining with sense probe. Comparing FIGS. 3 and 4,it was clear that in the liver tissue of the hepatic ischemia model rat,there was good staining around the central veins (the hole portion ofFIG. 4) (expression of HSC70 was high), and staining became fainter withdistance (expression of HSC70 was falling). From this, it can be saidthat in respect of HSC70, ischemia exhibited an effect around thecentral nerves.

EXAMPLE 4

Using a TATase probe instead of a HSC70 probe, in situ hybridization wasperformed according to similar steps to those of Example 3. As a TATaseprobe, position 192–594 (403 bp) of TATase (NCBI GenBank Accession No.X02741) labeled with digoxigenin was used. Primer sequences used inpreparation of RNA probes are shown in Table 5 and in situ hybridizationconditions are as shown in Table 6.

Results for the control rat are shown in FIG. 5, and results for thehepatic ischernis model rat are shown in FIG. 6. Within the figures,Anti-sense shows the results of staining with antisense probe, and Senseindicates results of staining with sense prove. Comparing 5 and 6, itwas clear that in the liver tissue of the hepatic ischemia model rat,there was good staining around the central veins (the hole portion ofFIG. 6) (expression of TATase was high), and staining became fainterwith distance (expression of TATase was falling). From this, it can besaid that in respect of TATase, ischemia exhibited an effect around thecentral nerves.

Regarding Hsc70 and TATase, the expression level according to GENECHIP™of both genes in both the control group, ischemia processed groupexhibit no particular dominance. However, with in situ hybridization, itis clear that expression around the central vein increased as betweenbefore and after ischemia. Further, it was clear that this change wasmarkedly appearing due: to TATase. Thus, by combining GENECHIP™ and insitu hybridization, information regarding a greater number of genes canbe obtained.

EXAMPLE 5

An adult rat (male, Wister-Kyoto, 12-weeks old, purchased from SankyoLab Service) whose bilateral body temperature and brain temperature wasmaintained at 37 degree. C., whose common carotid arteries werebilaterally ligated for 10 minutes to interrupt blood flow, and furtherwhose blood pressure was reduced to 30 to 40 mmHg, was adopted as abrain ischemia model (generally known as, Smith's brain ischemia model).After 10 minutes, reperfusion was allowed, and brain temperature andbody temperature were maintained at 37° C. After 2 hours, the rat waseuthanized, the hippocampus removed and a sample prepared following theprotocol. Using rat U34 array, gene expression analysis was perform withGENECHIP™ system of Affymetrics, Inc. (United States). With a rat U34array, it was possible to analyze 34,000 types of genome simultaneously.FIG. 7 shows results of analysis using bioinformatics (specifically, ascatter plot by LIMS-EDMT was used) on the basis of this data. FIG. 7,is a figure showing a scatter plot in respect of change in 34000 genesin a control group and 2 hours after ischemia. The horizontal axis ofFIG. 7 shows the genome expression level of a rat (control) in whichischemia processing was not conducted. The vertical axis indicates thegenome expression level 2 hours after ischemia/reperfusion. Theindividual points in Figure correspond to respective specified genomes.Examples of these include HSC70, HSP70, c-jun EST1 and EST2. Pointslying on the X-Y line indicate that there was no change in thecorresponding genes between before and after processing. As a result,comparing between 0 hours and 2 hours, there were approximately 475types where gene expression had increase 2 times or more, andapproximately 486 types where gene expression had fallen to ½ or less.In respect of HSC70, there was no change between before and afterischemia. (Intensity change Control*OH: approximately 30,000ischemia*2H: approximately 30,000). HSP70 exhibited an increase inexpression of as much as 20 times as between before and after ischemia(Intensity change Control*OH: approx. 1,000*ischemia*2H: approx.20,000). c-jun was hardly expressed at all prior to ischemia but afterischemia there was a dramatic increase in the expression thereof(Intensity change Control*OH: approx. 0.1→ischemia*2H: approx. 20,000).EST 1 was hardly expressed at all prior to ischemia but after ischemiathere was a dramatic increase in the expression thereof (Intensitychange Control*OH: approx. 3,000→ischemia*2H: approx. 15,000). EST2exhibited an increase in expression of as much as 5 times as betweenbefore and after ischemia (Change in intensity Control *OH: approx.3,000→ischemia*2H: approx. 15,000).

EXAMPLE 6

Brains were removed respectively from a control rat and the brainischemia model rat of Example 5 (after 2 hours had passed since recoveryof blood flow), and fresh frozen slices were prepared.Digoxigenin-labeled position 1182–1659 (478 bp) of HSP70 (Heat shockprotein 70 like protein, NCBI GenBank Accession No. L16764) was used asan RNA probe and in situ hybridization of brain fresh frozen slices wasperformed. Primer sequences used in preparation of RNA probes are shownin Table 5 and in situ hybridization conditions are as shown in Table 6.

Results for the control rat and brain ischemia model rat are shown inFIG. 8. Within the figures, anti-sense shows the results of stainingwith antisense probe, and sense indicates results of staining with senseprobe. Diffuse expression of HSP70 in the whole brain had increased. Inparticular, expression thereof increased markedly in the hippocampus.This matched with GENECHIP™ data and therefore provides support for thedata obtained with GENECHIP™.

EXAMPLE 7

Using c-jun probe instead of HSP70 probe, in situ hybridization wasperformed according to steps similar to those of Example 6. As a c-junprobe, digoxigenin-labeled position 453–878 of c-jun (426 bp) (NCBIGenBank Accession No.X17163) was used. Primer sequences used inpreparation of RNA probes are shown in Table 5 and in situ hybridizationconditions are as shown in Table 6.

Results for the control rat and the brain ischemia model rat are shownin FIG. 9. Within the figures, anti-sense shows the results of stainingwith antisense probe, and sense indicates results of staining with senseprobe. Diffuse expression of c -jun in the whole brain was increasing.This, in respect of the point that there had been no expression, but asa result of ischemia, there was a dramatic increase, matched withGENECHIP™ data and therefore provides support for the data obtained withGENECHIP™.

EXAMPLE 8

Using an EST1 probe instead of an HSP70 probe, in situ hybridization wasperformed according to steps similar to those of Example 6. As an EST1probe, digoxigenin-labeled position 147–511 (365 bp) of EST1 (NCBIGenBank Accession No.AA818604) was used. Printer sequences used inpreparation of RNA probes arc shown in Table 5 and in situ hybridizationconditions are as shown in Table 6.

Results for the control rat and the brain ischemia model rat are shownin FIG. 10. Within the figures, anti-sense shows the results of stainingwith antisense probe, and sense indicates results of staining with senseprobe.

Further magnified views of the results for the control rat and the brainischemia model rat are shown in FIG. 11 .

For EST1, deep staining was recognized in the hippocampus of the brainat low magnification in the dentate gyros and choroids layer of thebrain ventricle. Viewing with a microscopic magnification of 250x, 500x,it was clear that there was no expression prior to ischemia but afterischemia, cells of the internal skin of blood vessels were markedlystained. In this way, it is possible not only to specify differences inbrain distribution, but also differences in cell type within a tissue.

EXAMPLE 9

Using an EST2 probe instead of an HSP70 probe, in situ hybridization wasperformed according to steps similar to those of Example 6. As a EST2probe, digoxigenin-labeled position 183–585 (403 bp) of EST2 (NCBIGenBank Accession No. AI103915) was used. Primer sequences used inpreparation of RNA probes are shown in Table 5 and in situ hybridizationconditions are as shown in Table 6.

Results for the control rat and the brain ischemia model rat are shownin FIG. 12. Within the figure, anti-sense shows the results of stainingwith antisense probe, and sense indicates results of staining with senseprobe. For EST2, deep staining of hippocampus cone cells, before andafter ischemia in the brain, suggesting an increase in expression.

EFFECT OF THE INVENTION

The method of screening genes according to the method present inventioninvolves the innovative approach of selecting a gene from the novelpoint of view of localization of a gene and/or expression sequence tagin the tissue or cells of an organists, and is useful in narrowing downon a target gene.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: Nucleotide sequence of an upstream primer targeting thesequence from position 229 to 629 of HSC70.

SEQ ID NO: 2: Nucleotide sequence of a downstream primer targeting thesequence from position 229 to 629 of HSC70.

SEQ ID NO: 3: Nucleotide sequence of an upstream primer targeting thesequence from position 1182. to 1659 of HSP70.

SEQ ID NO: 4: Nucleotide sequence of a downstream primer targeting thesequence from position 1182 to 1659 of HSC70.

SEQ ID NO: 5: Nucleotide sequence of an upstream primer targeting thesequence from position 192 to 594 of TATase.

SEQ ID NO: 6: Nucleotide sequence of a downstream primer targeting thesequence from position 192 to 594 of TATase.

SEQ ID NO: 7: Nucleotide sequence of an upstream primer targeting thesequence from position 453 to 878 of c-jun.

SEQ ID NO: 8: Nucleotide sequence of a downstream primer targeting thesequence from position 453 to 878 of c-jun.

SEQ ID NO: 9: Nucleotide sequence of an upstream primer targeting thesequence from position 147 to 511 of EST1.

SEQ ID NO: 10: Nucleotide sequence of a downstream primer targeting thesequence from position 147 to 511 of EST1 .

SEQ ID NO: 11: Nucleotide sequence of an upstream primer targeting thesequence from position 183 to 585 of EST2.

SEQ ID NO: 12: Nucleotide sequence of a downstream primer targeting thesequence from position 183 to 585 of EST2.

1. A method of screening to identify a gene whose function is unknownpreviously, as a target for drug development, which comprises: (a)examining the expression of mRNAs and/or expression sequence tags being,products of gene expression before and after an event by using ahigh-density oligonucleotide array, and making a scatter diagram showingchanges in expression levels of the mRNAs and/or expression sequencetags between before and after the event, (b) determining one or morespecific mRNAs and/or expression sequence tags whose expression haschanged in response to the event, from the results in the scatterdiagram and from databases searches, (c) for each of said one or moremRNAs and/or expression sequence tags whose expression has changed inresponse to the event, designing a probe that will specificallyhybridize with the mRNA and/or expression sequence tag (d) performing insitu hybridization of at least two types of tissues or cells of anorganism before and after the event by using the one or more probesdesigned in step (c), (e) examining the localization of the one or moremRNAs and/or expression sequence tags in the tissues or cells before andafter the event, (f) determining whether the localization of those mRNAsand/or expression sequence tags has changed in response to the event,and (g) identifying those mRNAs and/or expression sequence tags whoseexpression and localization have both changed in response to the eventas a target for drug development.
 2. The method according to claim 1,wherein the mRNA and/or expression sequence tag is expressed in culturedcells or tissue.
 3. The method according to claim 1, wherein the geneencoding the mRNA and/or expression sequence tag has been cloned.
 4. Themethod according to claim 1, wherein the localization of at least twodifferent mRNAs and/or expression sequence tags is determined in asingle screening of the tissue or cell.
 5. The method according to claim1, wherein the gene encodes a substance effective as a drug.
 6. Themethod according to claim 1, wherein the gene is related to a disease.7. The method according to claim 1 comprising, after step (g), the stepof determining the function of the gene.
 8. The method according toclaim 1, wherein the tissue or cell is collected from an organism at twoor more different points in time after occurrence of an event.
 9. Themethod according to claim 1 or 8, wherein the event is ischemia orcancer.